While proteins produced in prokaryotes, such as Escherichia coli, have no carbohydrate chain, proteins produced in eukaryotes, such as yeasts, fungi, plant cells or animal cells, have a carbohydrate chain or chains in many instances.
Carbohydrate chains involved in glycosylation are roughly classifiable into two main groups. One group includes N-linked or N-glycosylated carbohydrate chains bound to the asparagine (Asn) residue in proteins and the other includes O-linked or O-glycosylated chains bound to the serine (Ser) or threonine (Thr) residue in proteins.
N-glycosylated carbohydrate chains have a common basic core structure composed of five monosaccharide residues, namely two N-acetylglucosamine residues and three mannose residues, and are classified into three types: high mannose type, complex type and hybrid type (FIG. 1). A precursor to these asparagine-linked carbohydrate chains is the lipid intermediate (Glc-.sub.3 Man.sub.9 GlcNAc.sub.2 -PP-Dol) (FIG. 2) composed of dolichol, which is polyisoprenoid alcohol comprising 18 to 20 isoprene units, and a carbohydrate chain composed of two N-acetylglucosamine residues, nine mannose residues and three glucose residues and bound to said dolichol via pyrophosphoric acid.
The reaction route leading to the formation of the lipid intermediate is well known as the "dolichol phosphate cycle" (FIG. 3).
The carbohydrate chain portion of the lipid intermediate is transferred as a whole to the Asn residue in an amino acid sequence (N-glycosylation site), such as Asn-X-Ser/Thr, in a polypeptide chain under formation within the cisterna of the rough-surfaced endoplasmic reticulum (rER), whereby an N-glycoside linkage is formed. In the above sequence, X may be any amino acid other than proline (Pro). This reaction is known to be catalyzed by "oligosaccharyl transferase", a kind of membrane enzyme. Thereafter, the carbohydrate chain undergoes trimming and processing in the process of passing through the rER and Golgi body, whereby a carbohydrate chain of the high mannose, hybrid or complex type is worked up (FIG. 4). It is known that a number of glycosidases and glycosyl transferases are involved in the process of trimming and processing.
While high mannose type carbohydrate chains are often encountered in glycoproteins of animal or plant origin as well as in yeast and fungal glycoproteins, it is presumed that carbohydrate chains of the complex type are limited to glycoproteins of the animal origin.
N-Glycosylated carbohydrate chains are bound to the Asn residue in Asn-X-Ser/Thr (X being any amino acid other than Pro) in polypeptides, as mentioned above. However, many proteins contain an unglycosylated Asn-X-Ser/Thr sequence or sequences and the presence of this sequence does not always result in addition of a carbohydrate chain thereto. In fact, William J. Lennarz et al. suggest that the three-dimensional structure of a protein is important in inducing binding of a carbohydrate chain. Their suggestion is based on the finding that simple tripeptides having the sequence Asn-X-Ser/Thr and denatured proteins free of a complicatedly folded spatial structure such as natural proteins have are comparatively readily glycosylated enzymatically in vitro.
On the other hand, O-glycosylated carbohydrate chains are bound to the Ser or Thr residue in polypeptides via N-acetylgalactosamine, which is generally followed by galactose, sialic acid, fucose and N-terminal acetylgalactosamine [Suzuki et al.: Tanpakushitsu, Kakusan, Koso, 30, 513 (1985)]. Unlike the case of the above-mentioned N-glycosylated carbohydrate chains, it is believed that their synthesis does not involve the rER but is always conducted in the Golgi body [Johnson et al.: Cell, 32, 987 (1983)]. Also, unlike the case of the N-glycosylated there is no rule on the amino acid sequence required for glycosylation. It is known, however, that the tendency toward glycosylation increases when Pro occurs in the vicinity, for example in the sequences Pro-Thr/Ser, Thr/Ser-Pro and Thr/Ser-X.sub.1-3 -Pro (X being any amino acid) [Takahashi et al.: Proc. Natl. Acad. Sci. USA, 81, 2021 (1984)].
Many of the substantial biologic functions of carbohydrate chains in glycoproteins remain unknown. However, a number of investigations on glycoproteins have already revealed diverse functions of carbohydrate chains.
Firstly, it is known that carbohydrate chains stabilize proteins. Retardation in blood clearance is an example. It is known that human erythropoietin (having no asparagine-linked carbohydrate chain) produced by means of gene introduction into Escherichia coli or human erythropoietin enzymatically treated for carbohydrate chain elimination shows activity in vitro but undergoes rapid clearance and shows decreased activity in vivo [Dordal et al.: Endocrinology, 116, 2293 (1985) and Browne et al.: Cold Spr. Harb. Symp. Quant. Biol., 51, 693 (1986)]. In the case of human granulocyte macrophage colony stimulating factor (hGM-CSF), the natural form of which has two N-glycosylated carbohydrate chains, it is known that the rate of clearance from the rat plasma increases in proportion to the reduction in the number of carbohydrate chains [Donahue et al.: Cold Spr. Harb. Symp. Quant. Biol., 51, 685 (1986)]. The rate of clearance and the site of clearance vary depending on the carbohydrate chain structure as well. Thus, it is known that sialic acid-containing hGM-CSF undergoes clearance in the kidney while hGM-CSF after sialic acid elimination shows an increased rate of clearance and undergoes clearance in the liver. Furthermore, .alpha..sub.1 -acid glycoproteins differing in carbohydrate structure as biosynthesized in a rat liver primary culture system in the presence of different asparagine-linked carbohydrate chain biosynthesis inhibitors were examined for the rate of clearance from the rat plasma and for the rate of clearance from the rat perfusate. It was found that in both fluids the clearance rates were in the following order: high mannose type&gt;carbohydrate chain-deficient type&gt;hybrid type&gt;complex type (natural form) [Gross et al.: Eur. J. Biochem., 162, 83 (1987)]. As another example of stabilization, it is known that carbohydrate chains provide proteins with protease resistance. In the case of fibronectin, for instance, inhibition of carbohydrate chain formation by means of tunicamycin results in an increased rate of decomposition of the intracellular product protein, i.e. carbohydrate chain-deficient fibronectin [Olden et al.: Cell, 13, 461 (1987)]. It is also known that carbohydrate chain addition increases thermal stability and/or freezing resistance. Furthermore, it is known that carbohydrate chains contribute to increased solubility of proteins, for example in the case of erythropoietin or .beta.-interferon.
Carbohydrate chains are helpful for proteins to maintain their proper three-dimentional structure. In the case of the vesicular stomatitis virus membrane-bound glycoprotein, it is known that removal of the two naturally occurring N-glycosylated carbohydrate chains results in inhibition of the transport of the protein to the cell surface and that addition of new carbohydrate chains to the protein results in recovery of this transport. In this case, it has been revealed that carbohydrate chain elimination leads to induction of the aggregation of one protein molecule with another via disulfide bond and, as a result, protein transport is inhibited. It is considered that the newly added carbohydrate chains can inhibit this aggregation and maintain the proper three-dimentional structure of the protein, thus making the protein transport again possible. In the case mentioned above, it has been shown that the sites for new carbohydrate chain addition are considerably flexible. To the contrary, it has been found that carbohydrate chain introduction at some sites results in complete inhibition of the transport of the protein having natural carbohydrate chains [Rose et al.: J. Biol. Chem., 263, 5948 and 5955 (1988)].
Instances are also known where antigenic sites on polypeptides are masked by carbohydrate chains. For hGM-CSF, prolactin, interferon-.gamma., Rauschcer leukemia virus gp70 and influenza hemagglutinin, experiments using polyclonal antibodies or monoclonal antibodies to specific regions on peptides have led to the conclusion that the carbohydrate chains on these proteins inhibit the reaction with the antibodies. On the other hand, it is also known that carbohydrate chains in some proteins induce immune reactions. It is thus suggested that carbohydrate chains might play a dual role.
It is further known that carbohydrate chains themselves are directly involved in the expression of activity of glycoproteins in some instances. Examples are glycoprotein hormones, such as luteinizing hormone, follicle-stimulating hormone and chorionic gonadotropin.
Finally, involvement in recognition phenomena may be mentioned as an important function of carbohydrate chains. Many instances are known where carbohydrate chains are considered to be involved in cell-cell, protein-protein or cell-protein recognition phenomena. That different carbohydrate chain structures may be indicative of different sites of in vivo clearance is an example.
In the foregoing, mention has been made of the structures and functions of carbohydrate chains in glycoproteins. The means of analyzing the structures and functions of carbohydrates have advanced remarkably, making it possible to analyze physicochemical properties of carbohydrate chains bound to peptide skeletons from various viewpoints.
In particular, it deserves special mentioned that highly specific enzymes (exoglycosidases) eliminating monosaccharides one by one and glycopeptidases or endoglycosidases cleaving the site of binding to a peptide chain without damaging either the peptide chain or the carbohydrate chain are now available for use in detailed investigations as to the biological roles of carbohydrate chains. It is also possible to add one or more additional carbohydrate chains to proteins by using glycosyltransferase. It is further possible to add sialic acid to the end of a carbohydrate chain by using sialyltransferase. Techniques are well known for modifying the carbohydrate chain to be added by using various glycosyltransferase inhibitors or glycosidase inhibitors.
Although there are some cases where the techniques of carbohydrate chain addition were applied for the purpose of investigating the functions of carbohydrate chains, as in the case of the above-mentioned vesicular stomatitis virus membrane glycoprotein, such techniques have never been used for the production of improved polypeptides of high commercial value. Generally, many (physiologically active) polypeptides have undesirable properties; for example, they are readily cleaved with protease and their activity is reduced, heat treatment reduce their activity, they readily undergo clearance when administered to living bodies, and so forth. There have been no cases known as yet in which attempts have been made to increase protease resistance, thermal stability or stability in blood by modifying the amino acid sequences of such polypeptides and intentionally adding one or more new carbohydrate chains to the polypeptide of interest. The present inventors have developed a means of improving various properties of such polypeptides, as mentioned above, through the intentional addition of one or more new carbohydrate chains to such peptides.
Generally, many physiologically active polypeptides are disadvantageous in that their activity is readily reduced by cleavage with protease or upon heat treatment or that they readily undergo clearance when administered to living animals including humans. For instance, urokinase (hereinafter referred to as "UK") is converted to an inactive form upon exposure to a protease called thrombin. It is an important task to improve physiologically active polypeptides with respect to such properties.